| Objective:Autoimmune thyroiditis(AIT)can cause destruction of thyroid tissue,and make damage to other vital tissues organs,in which steroid responsive encephalopathy associated with AIT is gradually being concerned by multidisciplinaries.Patients usually have high levels of anti-thyroid peroxidase(TPO)and/or anti-thyroglobulin(Tg)antibodies.However,it is reported that thyroid autoantibody titers have no correlation with the severity of encephalopathy,it is only a disease marker rather than a direct risk factor.Currently,cross-autoimmune responses induced by the common antigens in thyroid and brain tissue are considered to be the most likely pathogenic mechanisms.It is reported that PDIA3 were bound to Aβin cerebrospinal fluid(CSF).They suggested that PDIA3 became bound to normally N-glycosylated Aβcould prevent plaque formation,resulting from the precipitation of Aβin Alzheimer’s disease(AD),so PDIA3 can be an important cellular neuroprotective protein against toxicity.Moreover,PDIA3 plays an important role in the hypothalamic-pituitary-thyroid axis.It is reported that elevated Anti-PDIA3 Abs have been found in several autoimmune diseases and anti-PDIA3 Abs provided the great inhibition of PDIA3 activity,which may leading to loss of their function,could result in aberrantly folded proteins and hence protein aggregation or unfolded protein within cells,and cause disorders of thyroid hormonal system and central nervous system(CNS).However,it is still unclear whether PDIA3is the target antigen in thyroid and brain tissue during the development of AIT,and whether it has effects on inducing the autoimmune injury in thyroid and brain function,which has not yet been reported.In this study,SAT animal model,Tg and PDIA3recombinant protein were respectively used to establish the experimental animal model to investigate above mentioned questions,and provide new targets for clinical prevention and treatment of AIT-related injuries,especially the brain damage.Methods:The male NOD.H-2h4 mice were randomly divided into two groups.Control group were given sterile water.SAT group were given 0.005%(50 mg/L)sodium iodide(NaI)in their drinking waterduring the study period.Mice were sacrificed at 12 weeks.The serum levels of TgAb,PDIA3Ab and the imflammatory infiltration degree in thyroid tissues were detected.8-week-old CBA/J female mice were randomly divided into EAT group and control group.Each of mice in EAT group was given murine Tg(mTg)200μg and Freund adjuvant,then challenged 2 weeks later to establish the EAT animal model.Control group were treat with the sterile distilled water and Freund’s adjuvant.Mice were sacrificed at 4 weeks after the last immunization.The serum levels of TgAb,PDIA3Ab and the imflammatory infiltration degree in thyroid tissues were detected.The expression and localization of PDIA3 in normal thyroid and brain tissues of adult mice were determined.5-week-old CBA/J female mice were randomly divided into PDIA3 group and control group.Each of mice in PDIA3 group was given PDIA3 recombinant protein 75μg and Freund adjuvant,then challenged 2 weeks later to establish the experiment animal model.Correlation tests were performed at 4 weeks,6 weeks,10 weeks,14 weeks,18weeks after the last immunization.The serum levels of TgAb,PDIA3Ab and the imflammatory infiltration degree in thyroid tissues were detected.The deposition of C3,MAC,the precence of Ig G and GFAP,CD45~+cells aggregation were detected.Serum detection of TT4 and TSH to evaluate thyroid function in mice.RT-PCR analysis of the mRNA expressions of IFN-γ,IL-4,IL17 and TGF-βmRNA in spleens.Morris water maze and long-term potentiation(LTP)tests to evaluate the learning and memory ability of mice.The microvascular ultrastructure of cerebral cortex and hippocampus was observed by transmission electron microscope.Expression of cytokines and chemokines in brain were detected by proteomic microarray.Results:1.Models of SAT and EAT were established successfully,PDIA3Ab IgG were all significantly increased in SAT and EAT group than those of control group(P<0.05),and the PDIA3Ab total IgG was positively correlated with TgAb(P<0.05).In EAT group the levels of IgG1,IgG2a were higher in above 4 subclasses than those of control group.2.The expression of PDIA3 was found in normal mice thyroid follicular epithelial cells,vascular endothelial cells and in neurons,astrocytes,vascular endothelial cells of brain tissue,while not in microglia.3.After PDIA3 recombinant protein immunization,serum levels of PDIA3 IgG and IgG1,IgG2a,IgG2b were significant higher than those of control mice(P<0.05),and their high levels could be maintained to 18 weeks after the last immunization;the levels of IgG2a were higher in above subclasses.4.The results of RT-PCR showed that the mRNA expression of IL-4,IL17A and TGF-βin PDIA3 group at 10 weeks after the last immunization were significantly higher than that in the control group(P<0.05).The mRNA expression of IL17A at 14 and 18 weeks after PDIA3 immunization was higher than that in the control group Significantly(P<0.05).5.Serum levels of TgAb total IgG have no difference between PDIA3 group and control group.Both the incidence of autoimmune thyroiditis and the inflammatory score for intrathyroidal mononuclear cell infiltration were not show any differences in PDIA3 group compared with those of the control group.6.Since the the last immunization after 10 weeks,there was significant of IgG deposition in the follicular epithelial cells and vascular endothelial cells of C3 and MAC-positive thyroid tissue in the PDIA3 group.7.Fluorescence confocal microscopy showed that the deposition of IgG might be PDIA3Ab.8.The expression level of Cleaved Caspase-3 in thyroid tissues of PDIA3 group was significantly higher than that in control group.The serum level of TSH but not TT4 in PDIA3 group was higher than that in control group(P<0.05).9.Morris water maze experiment showed that the PDIA3 mice gradually showed a decline in spatial learning and memory ability.Result of LTP also showed showed a decline in and memory ability at 14 weeks after the last immunization.10.SEM observation revealed that there was significant edema in the cortex and hippocampus microvascular of PDIA3 group mice at 10 weeks after the last immunization.11.In the PDIA3 group,there was significant of IgG deposition in the nerve cells and vascular endothelial cells of C3-positive cerebral cortex and hippocampus.What’more,the deposition of IgG might be PDIA3Ab,and the aggregation of macrophages was revealed in the cerebral cortex and hippocampus with C3 deposition.12.The number of apoptosis cells at 10 weeks after the last immunization in cortex area of PDIA3 group was significantly higher than that in control group.13.IL-5,IL-6,IL-13,IL-12p70,MIG,MIP-1αin PDIA3 group of brain tissue have increasing trends,but without significant difference.Conclusions:1.PDIA3 was an autoantigen that coexists in the thyroid and brain tissues of normal mice and could be recognized by PDIA3Ab,which was specifically present in serum of SAT,EAT mice.In EAT group,the levels of IgG2a were higher in above 4subclasses,which has relatively strong complement-fixing abilities.2.PDIA3-immunization damage the follicular epithelial cells and vascular endothelial cells through CDC,result in the reserve dysfunction of thyroids.3.PDIA3-immunization could induce CDC,and through MAC damage blood-brain barrier permeability and nerve cells,and eventually decline the spatial learning and memory function of brain changes. |
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